Pseudomonas aeruginosa (PA) is the third most common gram-negative etiology of bloodstream infections, and these infections are associated with a crude mortality rate of 39%. Despite their frequency and severity, PA bloodstream infections are relatively poorly understood compared to pneumonia, burn infections, and keratitis. To investigate the pathogenesis of PA bloodstream infections, we have used a mouse model in which the tail vein is injected with a library of barcoded bacteria. Our preliminary experiments yielded several unexpected findings. First, in approximately half of severely ill bacteremic mice, the PA bacteria found disseminated throughout the body were descendants of just a few bacterial cells, suggesting that only a small number of the PA in the initial inoculum persisted and disseminated to cause severe disease. Second, PA bacteria in the blood migrated through a tight bottleneck to the gallbladder, which was a protective niche that allowed for a small number of PA to replicate to extremely high numbers. From there, these descendants of just a few PA bacteria seeded the intestines, presumably by passing through the common bile duct. This finding is particularly interesting in the context of other reports suggesting that PA is capable of migrating from the intestines to the bloodstream. Together, these observations suggest the intriguing hypothesis that spread of PA from the bloodstream to the intestines and back to the bloodstream may generate a positive feedback loop in which the gallbladder serves as an amplifier of PA numbers. In this application, we propose to address this limitation and directly test our hypothesis by performing the following specific aims: (1) Characterize bacterial dynamics over the course of PA bloodstream infections. (2) Determine whether interventions that disrupt PA transit through the intestines improve outcomes in bloodstream infections. Completion of these aims has the potential to uncover novel pathogenic mechanisms that contribute to the poor outcomes observed in PA bloodstream infections. The impact of these studies is three-fold: (i) they may provide a rationale for examining the pathogenesis of bloodstream infections caused by bacteria other than PA; (ii) the knowledge gained may be used as a foundation and justification for costlier and more laborious studies in humans with PA bloodstream infections; and (iii) these studies may inform novel therapeutic interventions that lower the unacceptably high mortality rates currently associated with PA bacteremia.